This invention relates to expression vectors for transfecting eucaryotic cells to produce transformants having a high level of expression of a protein of interest.
Gillies et al disclose in Cell, Vol. 33, pp. 717-728, Jul., 1983 and in more detail in copending U.S. application Ser. No. 592,231 filed Mar. 22, 1984 now U.S. Pat. No. 4,663,281, the disclosure of which is incorporated herein by reference, that specialized animal cells of the type which produce large quantities of a secreted protein such as globulins, fibrinogen, albumin, etc. exploit a tissue specific enhancer element present in their genome near the promoter and the gene encoding the protein. These cellular enhancer elements significantly enhance expression of a gene when located on either the 5' or 3' side of the gene. The functioning of this type of cellular enhancer is independent of the enhancer's orientation, and its ability to promote transcription from either homologous animal gene promoters or heterologous viral promoters is observed even when the enhancer element is disposed 10 kilobases (kb) or further away from the promoter.
The above-referenced Gillies et al application discloses methods of isolating such cellular enhancer elements from the genomes of animal cells and methods of exploiting their function in the construction of vectors designed to achieve expression of specific genes. The vectors may be used to transfect cells derived from the same tissue type in which the enhancer's activity is displayed naturally to produce valuable proteins. Transformants containing recombinant DNA including a cellular enhancer element and recombinant transcription unit such as a promoter ligated to a cDNA are characterized by high levels of transcription and translation of the cDNA and therefore significant improvements in expression.
The construction of chimeric viral and/or plasmid vectors containing such cellular enhancer elements has confirmed that these elements stimulate transcription of genes and promoters 10,000 base pairs or more from either the 5' or 3' end of the enhancer segments. Since stable, replicatable vectors typically comprise no more than about 20,000 base pairs, after transfection the enhancer stimulates transcription not only of the gene encoding the protein of interest, but also the gene encoding the marker protein which is used in the isolation of cells which have successfully incorporated the recombinant DNA.
It has been discovered that this link between expression of the marker protein and the protein of interest can limit the expression of the protein of interest. Transformants containing the recombinant plasmid typically are selected by culturing the cells in the presence of one or more toxic components. Only those cells which have been transformed by the recombinant vector and which express appropriate levels of the marker protein survive. Transfected cells expressing too low a level of the marker protein will not survive. Equally important, transfected cells expressing too high a level of the marker protein may also not survive. This is because too high a level of the marker protein may interfere with the cell's normal metabolism. Thus, transformed cells expressing very high levels of the protein of interest may be difficult to obtain because the concomitant high expression of the marker protein may be lethal.
An ideal expression system would uncouple the expression of the marker protein from the enhanced expression of the protein of interest. With such a system, transformed cells expressing levels of the marker protein appropriate for survival could express higher levels of the protein of interest than was previously possible.